Browsing by Issue Date, starting with "2009-05"
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- Estudo comparativo da citotoxicidade e alterações citopatológicas induzidas pela microcistina-LR em linhas celulares renais e hepáticas (Vero e HepG2).Publication . Menezes, Carina; Alverca, Elsa; Dias, Elsa; Paulino, Sérgio; Sam-Bento, Filomena; Pereira, PauloMicrocystin-LR (MCLR) is a potent hepatotoxin produced by freshwater cyanobacteria, being responsible for acute and chronic hazards to animal and human health. Despite the increasing recognition of its toxic effects, the cellular basis of MCLR-induced toxicity is still poorly understood. In this work, morphological, ultrastructural and biochemical (MTT and Neutral Red-NR) analyses were performed to evaluate the effects of a semi-purified MCLR-containing cyanobacterial extract on Vero and HepG2 cell lines. Our results showed that cell viability decayed markedly after 24h of exposure to toxin concentrations greater than 25μg.ml-1 in both cell lines. The ultrastructural analysis revealed that at subcytotoxic MCLR doses, cells presented large cytoplasmic vacuoles, which were more prominent in HepG2. These vacuoles showed to be enriched in the LC3 protein, suggesting that autophagy is an early cellular response to MCLR. At higher doses, the specific staining Acridine Orange (AO) and Rhodamine-123 (Rh-123), demonstrated that lysosome destabilization precedes mitochondrial dysfunction. Besides, MCLR seemed to induce a decrease in the expression of the anti-apoptotic endoplasmic reticulum protein Grp94, particularly evident in HepG2 cells. These results suggest that MCLR-induced cytotoxicity involves a considerable crosstalk among several organelles and that despite some cell-specific features, the general cellular basis underlying this toxicity is common to both Vero and HepG2 cells.
- Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutationsPublication . Bourbon, M.; Duarte, M.A.; Alves, A.C.; Medeiros, A.M.; Marques, L.; Soutar, A.K.Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH.
- Facts and fiction of genetically engineered foodPublication . Batista, Rita; Oliveira, Maria MargaridaThe generation of genetically engineered (GE) foods has been raising several concerns and controversies that divide not only the general public but also the scientific community. The fear and importance of the new technology, as well as commercial interests, have supported many of the ongoing discussions. The recent increase in the number of GE foods approved for import into the European Union and the increasingly global commercial food trades justify revisiting the facts and fiction surrounding this technology with the aim of increasing public awareness for well-informed decisions. Techniques that have recently become available for assessing food quality and its impact on human health, as well as the wealth of scientific data previously generated, clearly support the safety of commercialized GE products.